Chromatography Forum

Hi,
I am working on insulin molecule. It is present in combination with gelatin in the sample. Injecting the sample solution (containing gelatin) into HPLC column resulted in change of column chemistry (loss of retention). Could any one suggest, are there any special filters available in the market to separate gelatin from the sample. I am using RP C18 column.

Thanks for your help.

This is a tough problem, since your analyte and the matrix are very similar.

My first attempt would be to run a flat gradient on a column designed for peptide separations. You will get the gelatin gunk somewhere throughout the chromatogram, but if you are lucky, the insulin is in a relatively unoccupied part of the chromatogram. Or you could do LC/MS if you have this tool at your disposal. Of course, you should use a column designed for peptide analysis, and not a random C18, and you should use standard conditions for peptide gradients (0.1% TFA with a Peptide Analysis Column, contact me for details).

Alternatively, you could examine if you can get a sufficient fractionation of the gelatine and the insulin using SPE. This requires a fair amount of homework, and precooked methods won't work for this problem. In order to develop the SPE method, you will need a functional analytical method for the insulin. My first inclination is to do 2-D SPE, but there may be other options.

I am of the opinion that this is not a simple problem due to the similarity of the analyte and the background gunk.

I wonder if the problem could be solved using a sized exclusion type guard column. If the molecular size disparity is sufficient perhaps you could resolve the two?

Good Luck!

Kelly Johnson

If what you want to do is quantitate the insulin, how about skipping the C18 altogether and trying affinity chromatography. A quick Google search on "affinity chromatography" and "insulin" turns up several promising hints.

What is the mwt of the gelatin?

As gelatin is a protein of variable size MW approx 200,000 I should think it would be amenable to protein precipitation techniques eg Acetonitrile. Gelatin will definitely precipitate if mixed with any mobile phase containing organic solvent and ruin a column.

Hi,
As UWE suggested,it will be good if i choose a peptide column instead of normal C18 column.(peptide column can withstand gelatin into it?)We developed a method for Insulin using C18 column( used API during method Development),But the actual problem came when injecting sample with gelatin.
Regarding the solid phase extraction( as one of us told in forum),Its a very lengthy procedure of activation,flushing,retention and washing of solid bed and I dont have much information abt the solvents that could suit my molecule.
Is there any choice,to get rid of gelatin without changing the HPLC method.

Thank u

Please describe the gradient that you are using! Maybe some trivial things can help...

Gelatine can come in many different qualities! We use fish gelatine in one of our products, and it has a molecular weight of about 20.000.

Our chromatograms also become a mess after direct injection, so we use a SEC column to separate the gelatine and our analyte (Mw :1000) - trap the analytes on a precolumn - separate them on RP column - UV detection. It is fully automated and it works beautifully! However it requires two pumps and a six port switching valve.

Hi,
Mobile Phase A: sodium sulphate,pH=2.3 : ACN=82:18
Mobile Phase B: sodium sulphate,pH=2.3 : ACN=50:50 and gradient program is as follows

T %A %B
0 70 30
36 70 30
40 10 90
45 10 90
46 70 30
50 70 30

???

Do I read this correctly? You are running isocratically until 36 minutes in 70/30. Then you run a linear gradient over 4 minutes to 10/90.

If this is indeed what you are doing, I am not surprised that all kinds of things overlap with your insulin peak.

Imtakt has new applications now for the direct injection of gelatin on
Cadenza HS-C18.

Indomethacin in a gelatin aqueous solution:
http://www.imtakt.com/TecInfo/TI386E.pdf

Here, the gelatin is excluded and the inomethacin is retained.
Also - after 300 injections, there is no noticeable change in efficiency /
column back pressure

Calcitonin in gelatin aqueous solution:
http://www.imtakt.com/TecInfo/TI387E.pdf

If the mwt. of the gelatin in your capsule is > 30 kDa, it should be excluded when
injected on Cadenza HS-C18.

Insulin also gets nice peak shape on Cadenza HS-C18.